Cell Biology: Opening up new fronts in the fight against cholesterol

  1. Russell A DeBose-Boyd  Is a corresponding author
  2. Jay D Horton  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States

Since its discovery in 2003, the secretory protein PCSK9 has been the subject of growing interest because of its role in the degradation of low-density lipoprotein (LDL) receptors in the liver (Seidah et al., 2003). LDL receptors, which reside on the surface of liver cells, help to control cholesterol levels in the body by binding to low-density lipoprotein particles circulating in the blood and mediating their entry into the cells (Horton et al., 2009). Once inside the cells, the particles are released inside endosomes and are subsequently degraded in lysosomes, while the LDL receptors return to the cell surface to capture more low-density lipoprotein particles. PCSK9—which is short for proprotein convertase subtilisin-like type 9—prevents the LDL receptors from doing this job by binding to them and then diverting them to lysosomes for degradation. The physiological relevance of this reaction was first revealed by genetic analyses of humans with abnormal levels of lipids in their blood (Abifadel et al., 2003; Cohen et al., 2006), and confirmed in experiments using genetically manipulated mice (Maxwell and Breslow, 2004; Rashid et al., 2005; Lagace et al., 2006).

Gain-of-function mutations in PCSK9 lead to high levels of low-density lipoproteins in the blood (hypercholesterolemia) because they promote the degradation of LDL receptors, whereas loss-of-function mutations markedly reduce the levels of low-density lipoproteins. Since high levels of these lipoproteins are an important risk factor for atherosclerosis and associated coronary heart disease, finding ways to inhibit PCSK9 has become the focus of much research. Indeed, the results of recent clinical trials show that antibody-mediated inhibition of PCSK9 can reduce the levels of low-density lipoproteins in patients with hypercholesterolemia by as much as 65–70% (King, 2013). Now, in eLife, David Ginsburg of the University of Michigan and co-workers—including Xiao-Wei Chen as first author—report important insights into the transport of PCSK9 within cells (Chen et al., 2013).

PCSK9 is synthesized as a precursor in the endoplasmic reticulum (ER; Figure 1) and, like many other secretory proteins, it is subjected to post-translational modifications including glycosylation, phosphorylation, and tyrosine sulfation. Most studies to date have focused on PCSK9 once it has been secreted from cells. However, Chen and co-workers—who are based at Michigan, UC Berkeley, Wayne State University, the Cleveland Clinic and UCLA—have focused their attention on the secretion of PCSK9, and made the surprising discovery that decreased secretion of PCSK9 results in higher levels of LDL receptors, with a protein called Sec24A having a central role in the connection between the two.

Transport of the secretory protein PCSK9 from the endoplasmic reticulum (ER) to the Golgi.

When newly synthesized PCSK9 (shown in red) reaches the lumen of the endoplasmic reticulum (ER), it undergoes autocatalytic cleavage (1): This creates a prodomain that remains associated with the PCSK9 as it is exported from the ER and transported to the Golgi. Data from Chen et al. indicate that PCSK9 associates with a putative transmembrane receptor (shown in blue) that links it to a Sec23/Sec24A complex in the cytosol (2). This link is likely mediated by specific interactions between one or more sorting signals in the cytosolic domain of the receptor and binding sites in Sec24A. The receptor (along with PCSK9) is then incorporated into COPII-coated vesicles (not shown) for transport to the Golgi (3) and subsequent secretion. The Sar1 enzyme that triggers the formation of the vesicle, and its release from the ER membrane (thick black line), is also shown.

Proteins are transported from the ER to the Golgi by vesicles coated with coat protein complex II (COPII), which was first identified in yeast and consists of the enzyme Sar1, and complexes made of Sec proteins (notably the heterodimeric Sec23/Sec24 complex, and the heterotetrameric Sec13/Sec31). Sec24 mediates the packaging of the protein to be transported (which is called the cargo protein) into COPII-coated vesicles, and its importance is highlighted by the observation that deletion of the Sec24 gene in yeast cells results in their death. Mammals express four paralogs or versions of Sec24—Sec24A, Sec24B, Sec24C and Sec24D—and the deletion of these paralogs in mice results in phenotypes that range from a severe neural closure defect when Sec24B is deleted, to early embryonic lethality when Sec24D is deleted (Zanetti et al., 2012). It seems likely, therefore, that each paralog mediates the inclusion of specific cargo proteins into vesicles for subsequent export from the ER.

The cargo proteins transported by COPII-coated vesicles include transmembrane proteins that span the ER membrane one or more times, as well as soluble proteins that are contained entirely within the lumen of the ER. Biochemical and crystallographic studies indicate that incorporation of a particular transmembrane cargo protein into the transport vesicle is mediated by a binding site on the Sec24 protein, which is in the cytosol, and a specific signal presented by the cargo protein (Mancias and Goldberg, 2008). How soluble proteins became incorporated into transport vesicles is not completely understood. According to the ‘bulk flow’ model, all soluble proteins become incorporated into COPII vesicles by default without selection. However, incorporation of some soluble proteins into transport vesicles is mediated by their binding to transmembrane receptors that present specific sorting signals to the Sec24 proteins in the cytosol (Figure 1).

The results of Chen and co-workers indicate that the latter of these two scenarios applies to the export of PCSK9 from the ER. They find that mice deficient in Sec24A are remarkably normal in terms of survival, development and fertility. However, characterization of these mice also led to an unexpected discovery: a deficiency of Sec24A causes abnormally low levels of low-density lipoproteins in the blood (hypocholesterolemia) as a result of elevated levels of LDL receptors in the liver.

At least three lines of evidence indicate that these high levels of LDL receptors result from decreased secretion of PCSK9. First, the levels of PCSK9 in plasma of Sec24A-deficient mice are reduced compared to wild type animals. This is accompanied by increased levels of PCSK9 within liver cells and increased expression of LDL receptors on the surface of liver cells. Second, Sec24A binds to PCSK9, even though Sec24A is a cytosolic protein and PCSK9 is confined within the lumen of the ER (Figure 1). Third, overexpression of Sec24 promotes secretion of PCSK9, whereas reducing Sec24A expression by RNA interference-mediated knockdown blunts packaging of PCSK9 into COPII vesicles.

The action of Sec24A appears to be restricted to a subset of proteins that includes PCSK9. The activation of membrane-bound transcription factors called SREBPs requires their transport from the ER to the Golgi to be mediated by the ‘escort’ protein Scap (Brown and Goldstein, 2009). Chen et al. find that SREBP activation (and thus its Scap-mediated transport from the ER to the Golgi), continues normally in the livers of Sec24A deficient mice. This is consistent with the finding that Sec24C mediates the incorporation of the Scap protein into COPII vesicles (Sun et al., 2007).

Despite these observations, enthusiasm for strategies that reduce the secretion of PCSK9 by inhibiting Sec24A should be tempered until other proteins that require Sec24A for secretion are identified. The work of Chen et al. suggests that it might be better to inhibit the putative receptor that links PCSK9 in the ER to Sec24A in the cytosol (Figure 1). Identifying this receptor and elucidating how it works will be important for two reasons: it will teach us more about the export of PCSK9 and other soluble proteins from the ER in mammals, and it might lead to the development of novel therapies to reduce the levels of low-density lipoproteins in the blood and therefore help prevent atherosclerosis and heart disease.

References

Article and author information

Author details

  1. Russell A DeBose-Boyd

    Department of Molecular Genetics and the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Russell.Debose-Boyd@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Jay D Horton

    Departments of Internal Medicine and Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Jay.Horton@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.

Publication history

  1. Version of Record published:

Copyright

© 2013, DeBose-Boyd and Horton

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 597
    views
  • 110
    downloads
  • 2
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Russell A DeBose-Boyd
  2. Jay D Horton
(2013)
Cell Biology: Opening up new fronts in the fight against cholesterol
eLife 2:e00663.
https://doi.org/10.7554/eLife.00663

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Qian Wang, Jinxin Liu ... Qian Liu
    Research Article

    Paramyxovirus membrane fusion requires an attachment protein for receptor binding and a fusion protein for membrane fusion triggering. Nipah virus (NiV) attachment protein (G) binds to ephrinB2 or -B3 receptors, and fusion protein (F) mediates membrane fusion. NiV-F is a class I fusion protein and is activated by endosomal cleavage. The crystal structure of a soluble GCN4-decorated NiV-F shows a hexamer-of-trimer assembly. Here, we used single-molecule localization microscopy to quantify the NiV-F distribution and organization on cell and virus-like particle membranes at a nanometer precision. We found that NiV-F on biological membranes forms distinctive clusters that are independent of endosomal cleavage or expression levels. The sequestration of NiV-F into dense clusters favors membrane fusion triggering. The nano-distribution and organization of NiV-F are susceptible to mutations at the hexamer-of-trimer interface, and the putative oligomerization motif on the transmembrane domain. We also show that NiV-F nanoclusters are maintained by NiV-F–AP-2 interactions and the clathrin coat assembly. We propose that the organization of NiV-F into nanoclusters facilitates membrane fusion triggering by a mixed population of NiV-F molecules with varied degrees of cleavage and opportunities for interacting with the NiV-G/receptor complex. These observations provide insights into the in situ organization and activation mechanisms of the NiV fusion machinery.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Raji E Joseph, Thomas E Wales ... Amy H Andreotti
    Research Advance

    Inhibition of Bruton’s tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders, and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib, and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph et al., 2020). Here, we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.